Fig. 3: p53-mediated induction of PUMA ablates the MCL-1 block following IR or TMZ therapy.
A Immunoblots of MCL-1, PUMA and Noxa expression 48 hours post IR (5 Gy) or TMZ (50 µM) treatment, in 4 out the 20 gliomasphere lines assessed, all blots shown in Supplementary Fig. 3A. Dot plot of quantified immunoblots across p53 WT (n = 14) and mut-p53 (n = 6) gliomaspheres. One sample t test compares if p53 WT or mut-p53 changed relative to their vehicle gliomaspheres for all proteins assessed. Colors represent -log10 transformed p-values and dot sizes represent log2 transformed fold changes. log2 fold changes less than or equal 0.06 were set as the minimum for point sizes, and maximum -log10 p-value was set to 4 (p-value = 0.0001). For full immunoblots of all lines see Supplementary Fig. 3. These results were independently repeated. B PUMA expression in p53 KO models in GS025 and HK301, 48 hours post TMZ and IR treatment. C Immunoblots of GS025-shControl, shPUMA-1, and shPUMA-2, 48 hours after TMZ (50 µM) or IR (5 Gy) treatment. DBP of GS025-shControl, shPUMA-1, and shPUMA-2, 48 hours after TMZ (50 µM) or IR (5 Gy) treatment, shows change in precent cytochrome c release with HRK (100 µM) (mean ± s.d., unpaired t test with Welch correction, n = 2 biological replicates). D Apoptosis (Annexin V/PI + ) was evaluated in GS025-shControl, shPUMA-1, and shPUMA-2, 5 days after treatment with A1155463 (BCL-XLi: 0.5 µM), TMZ (50 µM), IR (5 Gy), TMZ + A1155463, or IR + A1155463 (mean ± s.d., unpaired t test with Welch correction, n = 3 biological replicates). E Co-immunoprecipitation of p53 WT gliomaspheres of BCL-XL or MCL-1 with PUMA following TMZ or IR. Gliomaspheres were treated for 48 hours with TMZ (50 µM) or IR (5 Gy). Signal quantification of PUMA determined relative to signal of MCL-1 or BCL-XL that was pulled down. These results were independently repeated. p > 0.05 = ns; p < 0.05 = *p < 0.01 = **p < 0.001 = ***p < 0.0001 = ****.
